Project 1: Rac1 induces PCK-dependent myosinIIA phosphorylation to regulate association with focal adhesions and cell migration. Pasapera AM1, Fischer RS1,Plotnikov SV1,Egelhoff T2, Waterman CM1. Cell Biology and Physiology Center, NHLBI, NIH1;Department of Cell Biology, Lerner Research Institute NC-10, Cleveland Clinic.2 Cell migration requires coordinated assembly of focal adhesions and contraction in the actomyosin cytoskeleton. The small GTPase Rac1 is critical to cell migration through its known functions in regulation of focal adhesion and actin cytoskeletal assembly dynamics, but its role in regulation of myosin II is not known. Myosin II dynamically assembles into minifilaments at the leading edge of migrating cells, and PKC-mediated phosphorylation in Ser 1916 in the non-helical tail is one of the main regulators. We hypothesized that Rac1 may regulate myosin II minifilament assembly dynamics during cell migration via downstream regulation of PKC and Ser 1916 phosphorylation. To test this, we analyzed the effects of Rac1 activation on the phosphorylation and dynamics of myosin IIA in U2OS cells. We found that transfection of active Rac1 (Rac1V12) induced PKC- and integrin-dependent myosin IIA phosphoryation on Ser 1916. Live cell imaging of GFP-myosin IIA revealed that Rac1 activation promotes rapid assembly, motion, and turnover of myosin IIA minifilaments, as well as perpendicular orientation to the leading edge, resulting in its accumulation specifically in focal adhesions. To determine the role of Ser 1916 phosphorylation on myosin IIA dynamics and localization, we expressed phospho-mimetic (S1916D) and non-phosphorylatable mutants (S1916A) of myosin IIA. This showed that phosphorylation is critical to the Rac1-induced rapid assembly and turnover of myosin IIA minifilaments as well as to the focal adhesion association of myosin IIA. Thus, Rac1 acts as a master regulator of cell migration by coordinating actin assembly-mediated protrusion, adhesion, and actomyosin contraction dynamics. This work was published in Current Biology Project 2:Mechanosensing by 2integrins regulates signaling and actin dynamics during phagocytosis Valentin Jaumouill Tissue resident phagocytes, such as macrophages and dendritic cells, act as sentinels of the immune system. They play a major role in the clearance of large particulate material, such as apoptotic cells and microbes. Depending on the nature of the particle they engulf, macrophages and dendritic cells will initiate an inflammatory response and present antigens to T lymphocytes. Phagocytosis depends on the reorganization of the actin cytoskeleton, driven by surface receptors. Although multiple signaling pathways have been identify, little is known about the molecular mechanisms underlying the formation of signaling complexes by the receptors, how actin reorganization is adjusted to the target biophysical properties, the mechanical forces involved in the uptake and whether they affect the downstream immune responses. We observed that engagement of 2 integrins by stiff particles lead to the assembly of a molecular platform that can act as a clutch to transduce mechanosensation. Whereas it has been previously established that 2-mediated phagocytosis of soft particles is independent of tyrosine kinases, we found that engulfment of stiff particles requires Src family kinases, Syk and the Arp2/3 complex. Live cell imaging reveals how target mechanical properties regulate actin dynamics in macrophages. This work will be presented at a Gordon conference and ASCB Project 4: YAP nuclear localization in the absence of cell-cell contact is mediated by a filamentous actin-dependent, myosin II- and phospho-YAP-independent pathway during ECM mechanosensing Arupratan Das YAP and TAZ transcriptional co-activators mediated up regulation of the target genes are responsible for development, tumor formation and cell differentiation. Nuclear exclusion of YAP through Hippo signaling mediated phosphorylation at S112 residue is well characterized. Actin being shown as common regulator of both Hippo signaling dependent as well as in Hippo independent regulation of YAP. Cellular perception of mechanical environment and regulation of YAP nuclear localization through actin is reported to determine differentiation fate. Here we showed that actomyosin contractility suppresses YAP phosphorylation at S112 residue however, neither loss of contractility nor increase in YAP phosphorylation is sufficient for its nuclear exclusion. Essential player for YAP nuclear localization is F-actin, which even triggers pS112-YAP nuclear localization independent of myosin contractility. Such actin mediated regulation is also conserved during mechanotransduction, as substrate compliance increased YAP phosphorylation and reduced F/G actin ratio leading to nuclear exclusion of both YAP and pS112-YAP. These data provide evidences for actomyosin contractility and phosphorylation independent regulation of YAP nuclear localization. In support to the physiological relevance, this study might help to explain reported observations where YAP dependent intestinal tissue growth persisted even in the activation of Hippo signaling and YAP phosphorylation at S112 residue This work was published in J. Biol. Chem. Project 4: Lindsay Case Focal adhesions (FAs) link the extracellular matrix (ECM) to the actin cytoskeleton to mediate cell adhesion, migration, mechanosensing and signaling. While FAs have conserved nanoscale architecture, it is unknown how the layered organization of proteins within FAs contributes to the regulation of protein activity and function. Vinculin is required for multiple FA functions, but it is unclear whether vinculins diverse protein interactions are spatiotemporally regulated. Using interferometric photo-activation localization (iPALM) super-resolution microscopy to assay vinculin nanoscale organization and a FRET biosensor to assay vinculin conformation, we found that upward repositioning within the laminar FA structure during FA maturation facilitates vinculin activation and mechanical reinforcement of FA. Inactive vinculin localizes to the lower integrin signaling layer by binding to phospho-paxillin. Talin binding activates vinculin and targets active vinculin to the higher FA layers where vinculin can engage retrograde actin flow. Thus, specific protein interactions occur within distinct FA nano-domains to regulate vinculin activation and function during FA maturation. This project resulted in 2 publications Project 5: Jeremy Logue and Richard Chadwick Abstract Within the confines of tissues, cancer cells can use blebs to migrate. Eps8 is an actin bundling and capping protein whose capping activity is inhibited by Erk, a key MAP kinase that is activated by oncogenic signaling. We tested the hypothesis that Eps8 acts as an Erk effector to modulate actin cortex mechanics and thereby mediate bleb-based migration of cancer cells. Cells confined in a non-adhesive environment migrate in the direction of a very large leader bleb. Eps8 bundling activity promotes cortex tension and intracellular pressure to drive leader bleb formation. Eps8 capping and bundling activities act antagonistically to organize actin within leader blebs, and Erk mediates this effect. An Erk biosensor reveals concentrated kinase activity within leader blebs. Bleb contents are trapped by the narrow neck that separates the leader bleb from the cell body. Thus, Erk activity promotes actin bundling by Eps8 to enhance cortex tension and drive the bleb-based migration of cancer cells under non-adhesive confinement. This project resulted in 3 publications